In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Branch has generated a knock-in murine model for OI with a classical collagen mutation. A fundamental insight into the mechanism of Oi derived from Brtl has involved the role of ER stress. Combined microarray and proteomic investigations of Brtl showed a two fold increase in Gadd153/CHOP expression and protein in lethal pups shortly before death. The increase in Gadd 153 expression was bone specific. Gadd153 is a member of the C/EBP family activated by cell stress, in particular, by ER retention of misfolded protein. These studies suggest that relief of ER stress with chemical chaperones, such as SPB, may have a beneficial effect on the OI skeleton. Using Brtl, we completed a major theraeputic trial of the effect of bisphosphonate, which complements our pediatric trial. In order to examine the effect of bisphosphonate on long bone (femur), we treated Brtl and wild-type littermates with alendonate and compared treated and untreated femora of each genotype. Alendronate treatment increased femoral DXA and cortical volumetric BMD, but did not improve Brtl weight curves or femoral length. Brtl trabecular number and diaphyseal cortical thickness were improved, as was femoral stiffness and load to fracture. However, detrimental changes were also detected in material and cellular parameters of bone. Predicted material strength and elastic modulus of both Brtl and wild-type bone were deceased;brittleness of Brtl femora were unchanged, while that of wild-type was increased. Furthermore, the material of the femora was changed by the dramatic retention of mineralized cartilage detected by light microscopy of Masson-stained sections, qCT attenuation and back-scatter EM. Embedded mineralized cartilage disrupts matrix continuity and may contribute to the weakening of bone material. In addition, the function of bone cells, although not their number, was impaired. Histomorphomnetry detected severe reductions in mineral apposition rate and bone formation rate. Osteoblast morphology was altered. Treated wild-type osteoblasts were more irregular in shape than the untreated cuboidal cells;Brtl treated osteoblasts have a flattened morphology, similar to lining cells. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. Brtl is currently being used for trials of two non-traditional therapies. In a collaborative study, Brtl was used for an in utero cell transplantation trial of GFP expression stem cells. Despite low levels of engraftment, the perinatal lethality and femoral geometry and biomechanics of the engrafted Brtl mice were improved. These results are encouraging for translational trials. Second, we are modelling a lesson from type I OI to suppress mutant collagen expression. Specific suppression of transcripts of the mutant collagen allele can biochemically transform individuals with severe OI into mild type I OI. We have introduced a RZ target site into the Brtl mutant allele;we have also generated transgenic mice expressing ribozymes targeted to the Brtl mutation. Preliminary data on Brtl/RZ mice is encouraging for improvement of Brtl biomechanical properties. BEMB has also collaborated in studies of the Old Order Amish mouse (OOA), a knock-in mouse with a glycine substitution in one Col1a1 allele at Gly610 to Cys. The human mutation occurs in a large Amish kinship with variability of expression. To compare the variability of expression in the mouse, the mutation was crossed into 4 different backgrounds and revealed changes crucial for bone strength and geometry. This approach can be useful for the identification of modifying factors for OI. We have identified a novel "high bone density" form of OI caused by mutation in the C-proteinase cleavage site. The Asp-Ala dipeptide between he telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. We have identified children with substitutions at two of these 4 peptides. They present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histomorphometry are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed. FTIR and BSEM are being used to study the amount and crystallinity of bone samples from our two probands. These data not only reveal a novel form of OI but also provide new fundamental information on roles of procollagen processing and the mechanism of tissue mineralization. To better understand the relationship of genotype and phenotype in human OI, the BEMB led and international consortium of connective tissue laboratories to assemble and analyze a mutation database containing over 830 mutations. Genotype-phenotype modeling revealed different functional relationships for each chain of type I collagen. Lethal mutations in alpha 1 (I) coincide with the Major Ligand Binding Regions. Lethal regions in alpha 2(I) continue to support the Regional Model first proposed by the BEMB, with lethal mutations in regularly-spaced clusters along the chain that coincide with proteoglycan binding regions. This model correctly predicts clinical outcome in 86% of alpha 2(I) mutations. <The consortium database now contains over 1300 mutations and the genotype-phenotype analysis will be updated in the coming year. We are also continuing our clinical studies of children with types III and IV OI. The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. However, the increment in vertebral BMD in the treatment group tapered off after one to two years of treatment.There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Hence the changes previously reported appear to have been a placebo effect in uncontrolled trials. We are recommending that treatment of children with types III and IV OI with pamidronate be limited to at most three years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial. We are also focusing on the variability of response to treatment in each group. The improvements in vertebral height and area do not correlate with changes in DXA z-score, nor did the improvement in vertebral height and area correlate for individual children. These differences may be related to important individual variation in ability to synthesize new bone or to remodel bone. They also highlight the inadequacy of DXA as a surrogate for bone strength.